Newly Described Coccidia Goussia Bayae From White Perch Morone Americana: Morphology And Phylogenetics Support Emerging Taxonomy Of Goussia Within Piscine Hosts Author Matsche, Mark A. Cooperative Oxford Laboratory, Oxford, Maryland 21654. Author Adams, Cynthia R. Cooperative Oxford Laboratory, Oxford, Maryland 21654. & National Fish Health Research Laboratory, Leetown Science Center, U. S. Geological Survey, Kearneysville, West Virginia 25430. Correspondence should be sent to Mark A. Matsche at: mark. matsche @ maryland. gov Author Blazer, Vicki S. Cooperative Oxford Laboratory, Oxford, Maryland 21654. & National Fish Health Research Laboratory, Leetown Science Center, U. S. Geological Survey, Kearneysville, West Virginia 25430. Correspondence should be sent to Mark A. Matsche at: mark. matsche @ maryland. gov & Cooperative Oxford Laboratory, Oxford, Maryland 21654. text Journal of Parasitology 2019 J. Parasitol. 2019-01-18 105 1 1 10 http://dx.doi.org/10.1645/18-67 journal article 10.1645/18-67 a39151be-8bb1-4dcb-bfcb-31af4e40f0f4 1937-2345 7752619 Goussia bayae n. sp. ( Figs. 1–4 ) Diagnosis: Oocyst shape: subspheroidal, with a single-layered smooth wall, ~ 0.4–0.6 thick; oocyst length (L) X width (W): 26.2 ± 2.1 SD X 21.8 ± 2.1 SD (ranges: 22–30 X 18–25); oocyst L/ W 1.2 ± 0.05 SD (range: 1.1–1.3) ( Figs. 1 , 2 ); micropyle present; oocyst residuum and polar granules absent; sporocyst shape: ellipsoidal, slightly tapered on 1 end, ~ 12.6 ± 0.6 SD X 7.8 ± 0.8 SD (ranges: 10–14 X 6–9), sporocyst L/ W 1.6 ± 0.1 SD (range: 1.4–1.9), with a smooth wall composed of 2 valves joined by a longitudinal suture; Stieda body, sub-Stieda body, sporocyst residuum absent; excysted sporozoite shape: slightly arcuate, 1 end tapered to a point; nucleus and refractile bodies present. Taxonomic summary Type host: White perch, M. americana (Gmelin 1789) ( Perciformes : Moronidae ). Other hosts: Unknown. Site of infection: Hepatic bile ducts, common duct, and gallbladder. Prevalence of infection: Of 150 fish examined, 100%. Type locality: Choptank River , Preston , Maryland ( 38°46 ′ 36.0 ′′ N , 75°58 ′ 10.4 ′′ W ) . Type specimens: Histological sections of infected tissues stained with H&E ( USNM 1507371 and 1507381) and photosyntypes ( USNM 1490787 , 1490788 , 1490789 , and 1490790) were deposited in the National Museum of Natural History , Smithsonian Institution , Washington , D.C. Etymology: The specific name is in honor of colleague and mentor Ana Baya. Remarks Phylogenetic analyses of other Goussia spp. using partial 18S rDNA placed G. bayae n. sp. in a clade of Goussia ( Fig. 5 ), defined as epicellular (sensu Rosenthal et al., 2016 ). Goussia bayae samples isolated from both the Choptank and Potomac were identical. The closest relative to G. bayae by Bayesian analysis was Goussia sp. isolate I29 (MF468318), despite sharing only 95.4% identity. The isolate is morphologically undescribed, but was found in the intestine of a farmed European bass ( Dicentrarchus labrax ) ( Xavier et al., 2018a ). The next nearest neighbors were also not fully morphologically described, but isolated from the heart and kidney of wild-caught chub mackerel Scomber japonicas (MF468319, MF468320), the spleen and kidney of the bluestripe snapper Lutjanus kasmira (HM117907), and the liver and spleen of the orange goatfish Mulloidichthys pfluegeri (HM117908). Goussia bayae shared 98.3% identity with Goussia sp. from the chub mackerel and 98.4% identity with sequences from the bluestripe snapper and orange goatfish. Of the remaining Goussia in the epicellular clade, only Goussia ameliae from alewives Alosa pseudoharengus has been reported from North America (Lovy and Friend, 2015). Figure 3. Light micrographs of coccidia of Goussia bayae n. sp. in the gallbladder of white perch, Morone americana . ( A ) Severe coccidiosis in gallbladder. Note that numerous oocysts were removed with the bile for differential interference contrast microscopy before histological processing. ( B ) Developing stages of coccidia along epithelium (arrow) and sporulating oocysts in the lumen. ( C ) Microgamont (Mi), macrogamont (Ma), and meront (Me) epicellular to biliary epithelium, with oocysts (O) in the lumen. Figure 4. Light micrographs of coccidia of Goussia bayae n. sp. in hepatic bile ducts of white perch, Morone americana . ( A ) Developing stages of coccidia epicellular to biliary epithelium. ( B ) Microgamont (Mi), macrogamont (Ma), and meront (Me) along epithelium. ( C ) Longitudinal view of bile duct with developing coccidia along epithelium (arrow) and sporulating oocysts (O) in lumen. ( D ). Cross-section of enlarged bile duct with numerous developing and mature coccidia. Figure 5. Bayesian phylogenetic relationships of fish-infecting Goussia and Choleoeimeria spp. based on partial 18S rDNA . Selected sequences represent different morphology types (epicellular, leucisci, dispersed, and nodular) defined by Rosenthal et al. (2016) . Hammondia hammondi was used as an outgroup. Posterior probability is indicated at branch sites. Accession numbers follow species names in parentheses. Goussia bayae possesses a micropyle ( Figs. 1 , 2 ), whereas the oocysts of all other epicellular species compared with here lack a micropyle and are distinctly smaller. The closest in size to G. bayae is G. ameliae (oocyst L X W: 18.6 X 14.1) from landlocked alewives and Goussia janae (oocyst L X W: 18.1 X 12.7). However, G. ameliae has abundant sporocyst residuum and G. janae sporocysts are longer (L: 13.5) and more slender (W: 5, L/W: 2.7) compared with the sporocysts of G. bayae . Goussia bayae is morphologically distinct from 2 eimerians previously reported from white perch. Oocyst diameters of E. glenorensis (12.0–10.5) and E. moronei (8.0–7.2) were markedly smaller than G. bayae and a micropyle was absent in both eimerians (Molnar and Fernando, 1974). Also, a Stieda body was present and refractile bodies were absent in E. glenorensi and E. moronei sporocysts. Histological examination of tissues indicated large numbers of coccidia in the gallbladder ( Fig. 3 ). Coccidian development was asynchronous and sporulation was endogenous. Meronts and gamonts were epicellular to the biliary epithelium, whereas oocysts were in the lumen ( Fig. 3B, C ). These stages of G. bayae were also observed in hepatic bile ducts and common duct ( Fig. 4 ). Meronts contained elongate merozoites that stained basophilic ( Figs. 3C , 4B ). Microgamonts contained microgametocytes that were scattered throughout the cytoplasm or aligned near the wall ( Figs. 3C , 4B ). Macrogamonts were larger than meronts and microgamonts, and contained a central eosinophilic nucleus and a coarse cytoplasm ( Figs. 3C , 4B ). Although prevalence was 100%, the severity of infection was highly variable among fish, ranging from mild to severe. In fish with mild infections, not all intrahepatic bile duct branches contained coccidia, whereas duct branches in severely infected fish were generally enlarged with numerous coccidia. Developing stages of G. bayae were not detected in the stomach, pyloric cecae, or intestine of fish. However, bile that passed through the ductus choledochus into the intestine contained sporulated oocysts of G. bayae . Figure 6. Bayesian phylogenetic relationships of partitioned 18S rDNA and cytochrome oxidase 1 ( COI ). Posterior probability is indicated at branch sites. Toxoplasma gondii served as an outgroup. Accession numbers follow species names in parentheses ( 18S rDNA , COI ). Molecular analyses also report the first mitochondrial DNA sequence from a Goussia species. The other evolutionarily distinct lineages of Goussia shown in Figure 5 do not yet have mitochondrial DNA sequences available. Bayesian analyses of the combination of gene markers 18S rDNA and COI ( Fig. 6 ), as well as of the partitioned mitochondrial genes ( COI and Cytb ) ( Fig. 7 ), definitively placed G. bayae basal to Choleoeimeria sp. and other Eimeria species with 0.999 bootstrap support. The nearest-neighbor by distance was Choleoeimeria sp. , which is similar in size, sporocyst, sporozoite, and excystation structures, and in biliary tissue localization, but infects reptiles ( Szczepaniak et al., 2016 ). In comparing mitochondrial genes, haematozoeans Babesia , Theileria , and Plasmodium are ancestrally related to the coccidians ( Fig. 7 ). Goussia bayae is sister group and basal to Eimeriidae , Choleoeimeria, and Sarcocystidae ( Fig. 7 ). Basal placement of G. bayae to Eimeriidae was also supported by analysis of all 3 mitochondrial genes (data not shown).